Molecular testing is emerging as a gold standard for some diagnostic tests due to their speed, sensitivity and specificity. Laboratory Developed Tests (LDTs) “are now one of the fastest growing segments in the in vitro diagnostic (IVD) market. Sample preparation is critical to the validity of the test, but frequently presents a bottleneck for clinical molecular biology workflows and diagnostic tests. While there are many molecular detection modalities, there are only a handful of automated sample preparation workflow strategies. Existing instruments that are built around these sample preparation strategies and chemistries range in cost from $17-$150 k, and yet they still do not provide an integrated method for processing difficult sample matrices such as raw sputum and/or difficult-to-disrupt organisms such as gram-positive bacteria and the acid fast bacilli (i.e., Mycobacterium).
Acid fast bacilli, including Mycobacterium strains are typically isolated from sputum of infected patients. They are known as “acid-fast bacilli” because of their lipid-rich cell envelope, which is relatively impermeable to various basic dyes unless the dyes are combined with phenol. Sputum is thick, viscous and difficult to process. Most sputum specimens for analysis contain various amounts of organic debris and a variety of contaminating, normal, or transient bacterial flora. Chemical decontamination/processing is typically used to reduce the viscosity and kill the contaminants while allowing recovery of the mycobacteria. Because of their unique cell envelope containing mycolic acids and a high lipid content, however, the cells are hydrophobic and tend to clump together. This renders them impermeable to the usual stains, such as the Gram stain. Two types of acid-fast stains are generally used, carbol fuchsin and fluorochromes, such as auramine or auramine-rhodamine. Once stained, the cells resist decolorization with acidified organic solvents, and are therefore called “acid-fast”. However, they retain fuchsine or auramine staining after successive or simultaneous treatment with acid and alcohol.
The sensitivity of acid-fast smear microscopy for Mycobacterium species is poor. The sensitivity of microscopy is influenced by numerous factors, such as the prevalence and severity of disease, the type of specimen, the quality of specimen collection, the number of Mycobacterium cells present in the specimen, the method of processing (direct or concentrated), the method of centrifugation, the staining technique, and the quality of the examination. It is recommended that a negative result should only be reported following the examination of at least 100 (in low-income countries) and preferably 300 (in industrialized countries) microscopic immersion view fields (or equivalent fluorescent view fields). Therefore, when microscopy is performed correctly, it can be time-consuming and laborious.
Mycobacterium strains are slow-growing bacilli, with a usual generation time of 12 to 18 hours. Colonies usually become visible only after a 1-week to 8-week incubation time. Samples which contain a low concentration of Mycobacterium cells further necessitate several subcultures. Mycobacterium cultures on specific media can allow for the identification of the particular Mycobacterium species contained in the biological sample. However, this is time-consuming, especially for those patients who are only at the beginning of the infection process.
Nucleic acid hybridization tests have been developed to detect strains of Mycobacterium in a biological sample. The first tests utilized direct probe hybridization. However, the concentration in Mycobacterium cells contained in a sample collected from a patient is usually too low to give a positive hybridization signal. Tests utilizing PCR amplification have therefore been developed. For example, the Gen-Probe® kit commercialized as “Amplified™ Mycobacterium tuberculosis direct test” kit, or MTD test kit, (Gen-Probe Inc., San Diego, Calif. 92121, USA) utilizes the amplification of MtbC-specific rRNA (Transcription-Mediated Amplification), followed by amplicon detection in accordance with the Gen-Probe HPA method (Hybridization Protection Assay).
In view of the above-described limitations, there is a need for simple and efficient systems integrating sample homogenization, lysis of difficult-to-disrupt microorganisms, and polynucleotide purification to meet the needs of both clinical laboratories and users alike.